In recent years, dozens of ADCs have been taken into preclinical and clinical development and two ADCs have been approved for marketing in the last couple of years. Apart from more recent developments for conjugating linker-drugs to (monoclonal) antibodies (mAbs), the drug in most of the ADCs in (pre)clinical development and in the two currently marketed ADCs is either linked to the antibody through the N-atom of a lysine residue or through the S-atom of a cysteine residue. The marketed product Kadcyla® or ado-trastuzumab emtansine (Roche/Genentech ImmunoGen) is an example of a lysine-linked ADC and Adcetris® or brentuximab vedotin (Seattle Genetics/Takeda Millennium) is an example of a cysteine-linked ADC. One of the ADCs currently in (pre)clinical development is a cysteine-linked ADC of formula (II) shown herein below in which a duocarmycin drug is conjugated through a cysteine residue to trastuzumab.
Duocarmycins, first isolated from a culture broth of Streptomyces species, are members of a family of antitumor antibiotics that include duocarmycin A, duocarmycin SA, and CC-1065. These extremely potent agents allegedly derive their biological activity from an ability to sequence-selectively alkylate DNA at the N3 position of adenine in the minor groove, which initiates a cascade of events that terminates in an apoptotic cell death mechanism.
In order to make Cys-linked ADCs, the antibody typically is partially reduced to convert one or more interchain disulfide bonds into two or more free cysteine residues. The thiol or sulfhydryl (SH) groups of the free cysteine residues are then subsequently conjugated with a linker-drug molecule to form a Cys-linked ADC. Typically, this conjugation process gives a random, heterogeneous mixture of antibodies loaded with 0, 2, 4, 6 and 8 linker-drugs. The lower is the average drug-to-antibody ratio (DAR), the higher is the amount of non-conjugated antibody (DAR0) in the reaction mixture.
Drug loading is known to have an effect on the antitumor activity of the ADC as described for example by K. J. Hamblett et al. in Clinical Cancer Research 10 (2004) 7063-7070. It also affects CMC (Chemistry, Manufacturing and Control) properties like aggregation.
WO2011/133039 of Applicant discloses a series of novel analogs of the DNA-alkylating agent CC-1065 and antibody-drug conjugates (ADCs) thereof. In Example 15, the preparation of a number of trastuzumab-duocarmycin conjugates has been described using 1.1 molar equivalents of a reducing agent to generate 2 free thiol groups per mAb. After quenching, the ADCs were purified using an r-Protein A column to give linker-drug conjugates having an average DAR of approx. 2.
The prior art discloses the use of hydrophobic interaction chromatography (HIC) as a polishing step in many monoclonal antibody (mAb) purification processes. It is mentioned that this mode of chromatography is particularly useful for aggregate removal, and it provides good clearance of other process-related impurities such as host cell protein(s), DNA, endotoxins, leached Protein A and endogenous viruses.
HIC is also a well-established method for the (analytical) determination of the DAR and drug load distribution for cysteine-linked ADCs (Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, 1045 (2013) 275-283). Chapter 17 of this book by Jun Ouyang depicts in FIG. 2 on page 276 a representative HIC chromatogram of a Cys-linked ADC (i.e., MC-VC-PABC-MMAE). It is mentioned that elution with a gradient of a decreasing salt concentration and an increasing organic modifier impacts the column retention of the drug-loaded species with the least hydrophobic, unconjugated form (i.e. non-conjugated antibody, DAR0) eluting first, and the most hydrophobic antibody with 8 linker-drugs (DARE) eluting last. The data in Table 2 on page 279 show that with a weighted average DAR of 3.6 the mixture of Cys-linked ADCs only contains 4.7% of non-conjugated antibody.
U.S. Pat. No. 4,771,128 describes a method for isolating and purifying toxin conjugates using HIC, in particular for immunoglobulin (antibody) conjugated to the toxic ribosome-inactivating protein ricin A. The method involves first removing unconjugated ricin A and aggregates via sizing chromatography (i.e., size exclusion chromatography, SEC), followed by hydrophobic gel chromatography (i.e., HIC, using Phenyl Sepharose CL-4B, volume 70 ml), in which the conjugate mixture was separated by eluting with salt solutions of decreasing ionic strength. The non-conjugated immunoglobulin was eluted first. The buffer used in both the sizing step and the subsequent chromatographic separation step contained sodium chloride (1 M) at a flow rate of about 20-40 ml/h, cf. Example 1. In an alternative embodiment, a “fast flow” chromatographic separation and purification is provided (i.e., using Phenyl Sepharose CL-4B, column diameter 1 cm, volume 3.14 ml) wherein the unconjugated immunoglobulin is removed with the first column volume of phosphate buffer/sodium chloride (1.5 M) solution at a flow rate of about 0.13 ml/h, cf. Example 2, and the immunoconjugate is removed with a second column volume of phosphate buffer containing 10-60 vol. % of an organic solvent (i.e., 60 vol. % glycerol in Example 2).
The main disadvantage of the methods disclosed in the prior art is the use of an organic solvent which is neither desirable nor acceptable for an industrial scale process.
A problem that has not been addressed in the prior art to the best of Applicant's knowledge is the scaling up of the ADC purification process.
Having reviewed the prior art, there is clearly a need for a new method for purifying mixtures of Cys-linked ADCs. In particular, it would be desirable to have a method for the purification of mixtures of Cys-linked ADCs having an average DAR of about 2-3, which typically contain a relatively high amount of non-conjugated antibody, sometimes as much as 40% by weight, on an industrial preparative scale, and not having to use multiple chromatographic steps.